Molecular chaperones maintain the appropriate folding and conformation of nascent polypeptides. This activity is crucial in regulating the balance between protein synthesis and degradation. When a protein is damaged, molecular chaperones may also facilitate their re-folding or, in the case of irreparably impaired proteins, their removal by the protein degradation machinery of the cell1 
Heat shock proteins (HSPs) were originally defined according to their increased expression in response to diverse cellular insults such as elevated temperature as well as exposure to heavy metals and oxidative stress1. Most, but not all HSPs are molecular chaperones that are organized into families according to their molecular size or function, including HSP100, HSP90, HSP70, HSP60, HSP40 and small HSPs. The rapid induction of HSP gene expression is referred to as the heat shock response (HSR) which confers cytoprotection to repeat exposure of the initial insult which would otherwise cause lethal molecular damage2. Cytoprotection is an example of increased molecular chaperone expression associated with the functioning of normal cells within an organism. However, aberrant expression of this family of proteins can also be associated with several disease states.
A large body of evidence exists supporting the role of molecular chaperones in maintaining the cancer phenotype. In addition, increasing evidence associating molecular chaperone expression with other disease including but not limited to: neurodegenerative disorders including Parkinson's, Alzheimer's, Huntington's and prion-related disease, inflammation and inflammation related disorders such as pain, headaches, fever, arthritis, asthma, bronchitis, tendonitis, eczema, inflammatory bowel disease, and the like, and diseases dependent on angiogenesis such as, cancer, arthritis, diabetic retinopathy, age associated macular degeneration (AMD) and infectious diseases in particular fungal infections, viral diseases including but not limited to diseases caused by hepatitis B virus (HBV), hepatitis C virus (HCV) and herpes simplex virus type-1 (HSV-1), cardiovascular and central nervous system diseases3,4,5,6,7.
HSP90 is an abundant molecular chaperone which constitutes 1-2% of total cellular protein. It exerts its chaperone function to ensure the correct conformation, activity, intracellular localization and proteolytic turnover of a range of proteins that are involved in cell growth, differentiation and survival3,5,8. Because of the large number of important signaling proteins with which HSP90 has been shown to associate and assist in stabilizing (these are generally called HSP90 client proteins), a rationale exists for the therapeutic use of HSP90 inhibitors for the treatment of a wide range of human diseases (as discussed above)9.
HSP90 activity is required for the stability and the function of many oncogenic client proteins, which contribute to all of the hallmark traits of malignancy, and thus, HSP90 has been widely acknowledged as an attractive therapeutic target for the treatment of cancer3,4,5,8. These client proteins include: BCR-ABL, AKT/PKB, C-RAF, CDK4, steroid hormone receptors (estrogen and androgen), surviving, c-Met, HER-2, and telomerases among others. Inhibition of HSP90 function leads to the destabilization and degradation of client proteins via the ubiquitin—proteasome pathway, resulting in the down-regulation of several signals being propagated via oncogenic signaling pathways and modulation of all aspects of the malignant phenotype3,5,8. Therefore, HSP90 inhibitors have potential to treat cancers driven by numerous diverse molecular abnormalities and their combinatorial effects could also reduce the possibility of resistance developing.
HSP90 is considered to exert its chaperone function via a cycle which utilizes the coordinated interaction of a number of co-chaperone proteins that are collectively involved in an orchestrated, mutually regulatory interplay with ATP/ADP exchange and ATP hydrolysis by the intrinsic and essential N-terminal ATPase domain. Crystallographic studies have revealed that several HSP90 inhibitors occupy the N-terminus ATP binding site10, thereby inhibiting HSP90 ATPase activity and function.
The 14-membered macrocyclic antibiotic radicicol was first demonstrated to have anti-tumor activity in vitro and shown to reverse the malignant phenotype of v-SRC transformed cells11. Subsequently, radicicol was shown using X-ray crystallography to bind to the N-terminal ATP-binding pocket of HSP90 with high affinity10, and to inhibit HSP90 ATPase activity resulting in the degradation of a number of signaling proteins12. Although radicicol inhibits tumor cell growth in vitro, it lacks activity in vivo, most likely due to its potentially reactive epoxide moiety and other adverse chemical features that cause instability and possible toxicity8,13.
The benzoquinone ansamycins are a second class of naturally occurring antibiotics which have been demonstrated to inhibit the activity of HSP90. The first example is geldanamycin which also competes with ATP for binding to the N-terminal nucleotide binding site of HSP9014. As was the case with radicicol, despite promising anti-tumor activity in vitro (and in vivo), the development of geldanamycin into a human therapeutic was stopped due to compound instability and unacceptable hepatotoxicity at therapeutic doses15.
Analogs of geldanamycin have been pursued with the objective of finding agents with an improved safety margin for clinical use, including the derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG or tanespimycin)16. 17-AAG has similar cellular effects to geldanamycin, including client protein degradation, and cell cycle arrest but with improved metabolic stability and lower toxicity5,8. Preclinical studies using 17-AAG have shown this derivative to be highly potent in vitro and to exhibit anti-tumor activity at non-toxic doses in various human tumor xenograft models17,18. Based on its biological activity, 17-AAG has recently completed several phase I clinical trials with some encouraging results9,19. As a result, 17-AAG has now entered phase II monotherapy clinical trials in various tumor types, including melanoma and breast.
There are several possible factors which may reduce the clinical efficacy of 17-AAG. Preclinical studies have shown that hepatic metabolism of 17-AAG by cytochrome P450 leads to the formation of 17-amino-17-demethoxygeldanamycin (17-AG)17. Although 17-AG retains inhibitory activity, metabolism by CYP3A4 is likely to be a cause of variable pharmacokinetics. In addition, the activity of 17-AAG is enhanced by its conversion to the hydroquinone form, 17-AAGH2, by the reductase enzyme NQO1 or DT-diaphorase17,20. The polymorphic expression of both of these metabolic enzymes may pose limitations for the clinical use of 17-AAG across the population5,8,17. The efficacy of 17-AAG may be further reduced by its association with the multi-drug resistance protein MDR1 or P-glycoprotein17. Finally, 17-AAG is limited by its poor solubility, cumbersome and complex formulation and lack of oral bioavailability. Attempts to reformulate 17-AAG have resulted in clinical trials commencing with CNF1010 and a cremaphore-based formulation (KOS-953) the latter of which has shown promising results during the phase I trial in patients with relapsed-refractory myeloma. The US National Cancer Institute and Kosan Biosciences have also developed a more water soluble and potentially orally bioavailable analog of 17-AAG, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG or alvespimycin, which was evaluated in preclinical and clinical trials9. 17-AAGH2, also known as IPI-504, has also entered clinical trial as a soluble derivative of 17-AAG21.
Non-natural product HSP90 inhibitors have recently been described. One of these contains a 3,4-diarylpyrazole resorcinol scaffold. These molecules were exemplified by Compound CCT018159, and analogues CCT0129397/VER-49009 and VER-50589. Treatment of cancer cells with these HSP90 inhibitors resulted in HSP70 induction, client protein depletion, cytostasis and apoptosis22,23,24,25.
Rational drug design was used by Chiosis et al.26 to develop a novel class of HSP90 inhibitors with a purine-scaffold. The first compound to be identified from this series, PU3, bound to HSP90 with moderate affinity resulting in cellular effects which are characteristic of HSP90 inhibitors26. An important feature of PU3 is that it is more soluble than 17-AAG; however, it is also significantly less potent against cells than the ansamycins26. Subsequent efforts focused on improving the potency of PU3 and led to the identification of PU24FC127. This compound exhibited biological effects on cells within a concentration range of 2-6 μM27, and also demonstrated 10-50 times higher affinity for HSP90 from transformed cells compared to that from normal tissues27. Administration of PU24FC1 in human breast tumor xenograft models led to anti-tumor activity without significant toxicity27. A more recent study has identified 8-arylsulfanyl, 8-arylsulfoxyl and 8-arylsulfonyl adenine derivatives of the PU class which exhibit improved water solubility and approximately 50 nM potency in cellular models, together with therapeutic activity in human tumor xenograft models28.
Additional non-natural product small molecule inhibitors of HSP90 have been identified including 2-amino-quinazolin-5-one compounds (WO2006113498A2), 2-amino-7,8-dihydro-6H-pyrido[4,3-d]pyrimidin-5-one compounds (WO2007041362A1) and quinazolin-oxime derivatives (WO2008142720A2) which target HSP90 for the prophylaxis or treatment of cell proliferative diseases. These molecules have reasonable potency and drug-likeness.
The preclinical proof-of-concept provide by small molecules with reasonable drug like properties coupled with the clinical proof-of-concept for the approach of inhibition of HSP90 activity achieved with 17-AAG, has generated a high level of interest in industry to develop additional HSP90 inhibitors with improved drug like properties that can provide therapeutic benefit to patients suffering from disease states related to abnormal protein folding.
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Stapleton, Y. Asad, L. Simmons, A. Maloney, F. Raynaud, M. Campbell, M. Walton, S. Lakhani, S. Kaye, P. Workman and I. Judson, Phase I pharmacokinetic and pharmacodynamic study of 17-allylamino, 17-demethoxygeldanamycin in patients with advanced malignancies, J. Clin. Oncol. 23 (2005), pp. 4152-4161.    20. W. Guo, P. Reigan, D. Siegel, J. Zirrolli, D. Gustafson and D. Ross, Formation of 17-allylamino-demethoxygeldanamycin (17-AAG) hydroquinone by NAD(P)H:quinone oxidoreductase 1: role of 17-AAG hydroquinone in heat shock protein 90 inhibition, Cancer Res. 65 (2005), pp. 10006-10015.    21. J. R. Sydor, E. Normant, C. S. Pien, J. R. Porter, J. Ge, L. Grenier, R. H. Pak, J. A. Ali, M. S. Dembski, J. Hudak, J. Patterson, C. Penders, M. Pink, M. A. Read, J. Sang, C. Woodward, Y. Zhang, D. S. Grayzel, J. Wright, J. A. Barrett, V. J. Palombella, J. Adams and J. K. Tong, Development of 17-allylamino-17-demethoxygeldanamycin hydroquinone hydrochloride (IPI-504), an anti-cancer agent directed against Hsp90, Proc. Natl. Acad. Sci. USA 103 (2006), pp. 17408-17413.    22. K. M. Cheung, T. P. Matthews, K. James, M. G. Rowlands, K. J. Boxall, S. Y. Sharp, A. Maloney, S. M. Roe, C. Prodromou, L. H. Pearl, G. W. Aherne, E. McDonald and P. Workman, The identification, synthesis, protein crystal structure and in vitro biochemical evaluation of a new 3,4-diarylpyrazole class of Hsp90 inhibitors, Bioorg. Med. Chem. Lett. 15 (2005), pp. 3338-3343.    23. S. Y. Sharp, K. Boxall, M. Rowlands, C. Prodromou, S. M. Roe, A. Maloney, M. Powers, P. A. Clarke, G. Box, S. Sanderson, L. Patterson, T. P. Matthews, K. M. Cheung, K. Ball, A. Hayes, F. Raynaud, R. Marais, L. Pearl, S. Eccles, W. Aherne, E. McDonald and P. Workman, In vitro biological characterization of a novel, synthetic diaryl pyrazole resorcinol class of heat shock Protein 90 inhibitors, Cancer Res. 67 (2007), pp. 2206-2216.    24. B. W. Dymock, X. Barril, P. A. Brough, J. E. Cansfield, A. Massey, E. McDonald, R. E. Hubbard, A. Surgenor, S. D. Roughley, P. Webb, P. Workman, L. Wright and M. J. Drysdale, Novel, potent small-molecule inhibitors of the molecular chaperone Hsp90 discovered through structure-based design, J. Med. Chem. 48 (2005), pp. 4212-4215.    25. S. Y. Sharp, C. Prodromou, K. Boxall, M. V. Powers, J. L. Holmes, G. Box, T. P. Matthews, K. M. Cheung, A. Kalusa, K. James, A. Hayes, A. Hardcastle, B. Dymock, P. A. Brough, X. Barril, J. E. Cansfield, L. Wright, A. Surgenor, N. Foloppe, R. E. Hubbard, W. Aherne, L. Pearl, K. Jones, E. McDonald, F. Raynaud, S. Eccles, M. Drysdale and P. Workman, Inhibition of the heat shock protein 90 molecular chaperone in vitro and in vivo by novel, synthetic, potent resorcinylic pyrazole/isoxazole amide analogues, Mol. Cancer. Ther. 6 (2007), pp. 1198-1211.    26. G. Chiosis, M. N. Timaul, B. Lucas, P. N. Munster, F. F. Zheng, L. Sepp-Lorenzino and N. Rosen, A small molecule designed to bind to the adenine nucleotide pocket of Hsp90 causes Her2 degradation and the growth arrest and differentiation of breast cancer cells, Chem. Biol. 8 (2001), pp. 289-299.    27. M. Vilenchik, D. Solit, A. Basso, H. Huezo, B. Lucas, H. He, N. Rosen, C. Spampinato, P. Modrich and G. Chiosis, Targeting wide-range oncogenic transformation via PU24FC1, a specific inhibitor of tumor Hsp90, Chem. Biol. 11 (2004), pp. 787-797.    28. H. He, D. Zatorska, J. Kim, J. Aguirre, L. Llauger, Y. She, N. Wu, R. M. Immormino, D. T. Gewirth and G. Chiosis, Identification of potent water soluble purine-scaffold inhibitors of the heat shock protein 90, J. Med. 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